Diagnostic agent and method for determining glucose

ABSTRACT

GLUCOSE IS DETERMINED ENZYMATICALLY WITH A STABLE DIAGNOSTIC AGENT MIXTURE COMPRISING HEXOLKINASE, GLUCOSE-6PHOSPHATE DEHYDROGENASE, ATP, MAGNESIUM SULFATE, NADP. AND A BUFFER CONTAINING AN AZIDE.

United States Patent O 3,778,350 DIAGNOSTIC AGENT AND METHOD FORDETERMINING GLUCOSE Hans Ulrich Bergmeyer, Tutzing, Upper Bavaria, ErichBernt, Munich, Wolfgang Gruber, Garatshausen, Felix Helmut Schmidt,Mannheim-Seckenheim, and Harald Stork, Lampertheim, Hesse, Germany,assignors to Boehringer Mannheim GmbH, Mannheim, Germany No Drawing.Filed Aug. 5, 1970, Ser. No. 61,503

Claims priority, application Germany, Aug. 11, 1969, P 19 40 816.3 Int.Cl. G01n 31/14 US. Cl. 195-103.5 C 8 Claims ABSTRACT OF THE DISCLOSUREGlucose is determined enzymatically with a stable diagnostic agentmixture comprising hexokinase, glucose-6- phosphate dehydrogenase, ATP,magnesium sulfate, NADP, and a buffer containing an azide.

The present invention is concerned with a diagnostic agent and with aprocess for the enzymatic determination of glucose by means ofhexokinase and glucose-6-phosphate dehydrogenase.

It is known to determine glucose enzymatically by converting it intoglucose-fi-phosphate with adenosine triphosphate and hexokinase. Theresultant glucose-o-phosphate is converted into gluconate-G-phosphate bymeans of nicotinamideadenine-dinucleotide-phosphate (NADP) in thepresence of glucose-6-phosphate dehydrogenase (G-6- PDI-I). In this way,there is formed an amount of reduced nicotinamide adenine dinucleotidephosphate (NADPH) which is equivalent to the amount of glucose-6-phosphate. The latter can very easily be determined photometrically.

This known process is very dependable, specific and essentially notsubject to disturbance and is especially useful for the determination ofglucose in biological fluids, as well as in foodstuffs. However, adisadvantage of this known process is that the solutions of theindividual reagents, namely, buffer, ATP, NADP, as well as magnesiumsulfate, must be separately pipetted because solutions of ATP in thepreviously used mixtures of triethanolamine hydrochloride, sodiumhydroxide or sodium carbonate and magnesium sulfate, are not stable and,after some time, show considerable turbidity.

We have now found that ATP, triethanolamine hydrochloride, sodiumhydroxide or sodium carbonate and magnesium sulfate can all be kepttogether in solution when an azide is added to the solution. Noturbidity then occurs.

Thus, the instant invention provides a novel diagnostic composition, notsubject to the inherent disadvantages of conventional diagnostic agents,as well as a novel method for determining glucose.

The method according to the present invention for the enzymaticdetermination of glucose by conversion into glucose-6-phosphate by meansof adenosine triphosphate (ATP) and hexokinase, oxidation thereof intoglucose-6- phosphate by means of nicotinamide-adenine-dinucleotidephosphate (NADP) in the presence of glucose-6 phosphate dehydrogenase(C-6-PDH), with the formation of reduced nicotinamide adeninedinucleotide phosphate (NADP) and spectrophotometric determination ofthe latter, is characterized by using a buffer which contains an azide.Preferably, there is used a buffer which comprises a mixture oftriethanolamine hydrochloride, sodium hydroxide or sodium carbonate andsodium azide.

By means of the method according to the present invention, it ispossible to bring together the reagents needed for the reaction, apartfrom the enzymes and NADP, and

ice

to use them in this form. Solutions which contain the ATP, magnesiumsulfate, sodium hydroxide or sodium carbonate, triethanolaminehydrochloride and sodium azide, show no turbidity and no decrease in theATP content even after storage for 3 months at 33 C., whereas thepreviously used solutions, which did not contain sodium azide, could, atmost, be kept in a refrigerator for up to 14 days. In the case of longerstorage of these known solutions, the concentration of the ATP dropped01f so considerably over time that, when using such a preparation,erroneous results are obtained.

The new diagnostic reaction mixture provided by the present inventioncan also be stored in solid form, as a homogeneous powder mixture, inairand moisture-tight containers at ambient temperature so that themixture can be supplied in this form. Surprisingly, the azide does notdisturb the enzymatic reaction.

Frequently, it is desirable, in the case of the method according to thepresent invention, also to add a surfaceactive substance, preferablydigitonin or Sterox (an ethylene oxide adduct produced by the MonsantoChemical Co.), to the solution of the reagents, the surface-active agentpreferably being used in an amount of about 10 mg./ ml. of reagentsolution.

The present invention also provides a diagnostic agent for the enzymaticdetermination of glucose, which comprises hexokinase andglucose-6-phosphate dehydrogenase, as well as adenosine-triphosphate(ATP), magnesium sulfate, nicotinamide adenine dinucleotide phosphate(NADP) and a buffer which contains an azide.

In the diagnostic agent according to the present invention, theadenosine-triphosphate, magnesium sulfate,nicotinamide-adenine-dinucleotide phosphate, buffer substance and azideand optionally also a surface-active agent, such as dignitonin, are madeup in the form of a homogeneous powder mixture, while the enzymeshexokinase and glucose-'6-phosphate-dehydrogenase are made up separatelyin the form of a suspension in an ammonium sulfate solution.

The buffer substances preferably acomprises triethanolaminehydrochloride and sodium hydroxide or sodium carbonate, together with anazide, such as sodium azide. Other buffers which can be used include,for example, tris-hydroxy-methylaminomethane/succinic acid andimidazole/succinic acid, together with an azide, such as sodium azide,the pH value of the buffer being between 6.8 and 7.5.

As azide, there can be used the alkali metal or alkaline earth metalazides, for example, sodium, potassium, lithium, magnesium or calciumazide, or also ammonium azide.

According to another embodiment of the diagnostic agent according to thepresent invention, the adenosine triphosphate, magnesium sulfate, buffermixture and azide and optionally also a surface-active agent, such asdigitonin, are present in the form of an aqueous solution, thenicotin-amide-adenine-dinucleotide phosphate is in solid form (forexample, pressed to form a tablet) and the enzymes hexokinase andglucose 6-phosphate dehydrogenase in the form of a separate suspensionin a solution of ammonium sulfate.

The diagnostic agent according to the present invention preferablycomprises 1-15% by weight magnesium sulfate heptahydrate, 0.5-6% byweight nicotinamideadenine-dinucleotide phosphate, 0.56% by weightadenosine triphosphate, 2.0-18% by weight sodium azide, 0.10-1.5% byWeight of a surface-active agent, such as digitonin, and 50-95% byweight of a buffer mixture, as well as the enzyme suspension.

The buffer mixture preferably comprises 1.56-1 1.0 parts by weighttriethanolamine hydrochloride and 0.6 -3.5 parts by weight sodiumcarbonate; or 1.2-7.5 parts by weight tris-hydroxymethylaminomethane and0.55-3.45 parts by weight succinic acid; or 0.745 parts by weightimidazole and 0.70-1.75 parts by weight succinic acid.

For carrying out the determination of glucose with the diagnostic agentaccording to the present invention, the diagnostic agent, if present inthe form of a powder mixture, is simply dissolved before use in anappropriate amount of distilled water. The amount of unused solutioncan, if desired, be kept for several weeks in a refrigerator withoutloss of quality. Subsequently, in the usual manner,. theglucose-containing sample, for example whole blood, is added and theextinction determined, whereupon the enzyme mixture of hexokinase andglucose-6-phosphate dehydrogenase is added and the extinction againmeasured. The enzyme suspension can be kept for years without specialprecautions, especially when stored in a refrigerator. The process anddiagnostic agent according to the present invention thus permit anextremely simple carrying out of the process for the determination ofglucose and avoid the necessity for having to pipette out separatelynumerous individual reagents for each determination and also of havingto prepare fresh solution repeatedly.

The superior stability of adenosine triphosphate in aqueous solutionachieved by means of the present invention can be appreciated from thecomparative experiments described hereinafter. In these experiments, anaqueous solution of the diagnostic agent according to the presentinvention, which contained adenosine triphosphate in the form of itsdisodium salt, magnesium sulfate, triethanolamine hydrochloride, sodiumcarbonate, digitonin and sodium azide, was compared with a solution madeup in the same manner but with the omission of the sodium azide. Thefollowing results were obtained:

Cone. of ATP, Decrease of ATP,

mg./ml. cone. in percent With Without With Without Time azide azideazide azide Commencement 0. 425 0. 450 After-- 10 days.- 0.425 0. 450 020 days.-- 0. 420 0. 170 -1. 2 --62 50 days- 0. 418 0. 040 --1. 6 91 90days 0.390 0. 000 --8 100 1 ATP=Adenosine triphosphate.

EXAMPLE 1 The following components were mixed together to form ahomogeneous powder:

Grams Triethanolamine hydrochloride 11.0 Sodium carbonate 3.5 Magnesiumsulfate heptahydrate 0.2 Nicotinamide-adenine-dinucleotide phosphate 0.1

Adenosine--triphosphate disodium salt trihydrate 0.1 Sodium azide 0.3Digitonin 0.02

150 mg. amounts of this mixture were weighed out into bottles which werethen closed in an airtight manner. The contents of a bottle were disolved before use by the addition of 2.0 ml. water and brief shaking upand then mixed with 0.0050.2 ml. of the sample to be investigated, suchas urine, blood, serum or the like. The extinction E was measured at 366nm. or at 340 nm. with a suitable photometer. Thereafter, there wasadded one drop of an enzyme suspension which contained about units ofhexokinase and 75 units of glucose-6-phosphate-dehydrogenase per ml. 3 Mammonium sulfate solution.

After about 5-10 minutes, a constant extinction value E was obtained.The extinction difference AE=E E was multiplied by a calculation factorand gave directly the concentration of glucose in the sample.

The unused solution can be kept for months and can be used again withoutalteration of the procedure.

By use of the digitonin-containing solution, it was possible to addblood directly to the test mixture without carrying out a previousdeproteinization or removal of the red blood corpuscles which interferewith an optical measurement. In the case of the present invention, only0.005-0.01 ml. of blood was needed.

EXAMPLE 2 2.5 ml. of a solution containing:

Grams Triethanolamine hydrochloride 0.140 Adenosine5'-triphosphate 0.020Magnesium sulfate heptahydrate 0.003 Sodium carbonate 0.016 Sodium azide0.0025 Digitonin 0.00025 were pipetted into a measurement cuvette, atablet of NADP was dissolved therein and 0.01 ml. whole blood was added.The haemolysis brought about by the digitonin present was finishedpractically immediately. Then, as described in Example 1, the extinctionE was measured and the reaction commenced with the enzyme mixture, as inExample 1. The measurement and evaluation took place as in Example 1.

What is claimed is:

1. Diagnostic agent for the enzymatic determination of glucose whichagent comprises two reagents:

(1) a first reagent consisting essentially of adenosine triphosphate,magnesium sulfate, nicotinamide-adenine-dinucleotide phosphate, a buffersubstance comprising triethanolamine hydrochloride, sodium hydroxide orsodium carbonate, and azide made up as a homogenous powder mixture freeof hexokinase and GPDH, and

(2) a second reagent consisting essentially of the enzymes hexokinaseand glucose-6-phosphate dehydrogenase made up separately in the form ofa suspension in a solution of ammonium sulfate, the second reagent beingfree of azide.

2. Diagnostic agent as claimed in claim 1 wherein the first agentadditionally contains a surface active agent.

3. Diagnostic agent as claimed in claim 2, consisting essentially of115% by weight magnesium sulfate, 0.5- 6% by weightnicotinamide-adenine-dinucleotide phosphate, 0.56% by weight adenosinetriphosphate, 2.0-18% by weight of an azide, 0.10-1.5% by weight of asurfaceactive agent and 50-95% by weight of a buffer.

4. Diagnostic agent as claimed in claim 1, wherein the azide used isammonium azide or an alkali metal or alkaline earth metal azide.

5. Diagnostic agent as claimed in claim 4, wherein the azige is sodium,potassium, lithium, magnesium or calcium az1 e.

6. Diagnostic agent as claimed in claim 2, wherein the surface activeagent is digitonin.

5 6 7. Process for the enzymatic determination of glucose Pearse:Histochemistry-Theoretical and Applied, which comprises contacting theglucose-containing matepp. 560-561 (1960). rial with a diagnostic agentas claimed in claim 1. Bergmeyer: Methods of Enzymatic Analysis, pp.117- 8. Process for the enzymatic determination of glucose 119 (1965).in serum which comprises contacting the glucose-contain- 5 ChemicalAbstracts, II, 39:5275 (1945); ibid., III, ing serum with a diagnosticagent as claimed in claim 1. 43:269i (1949).

References Cited ALVIN E. TANENHOLT Z, Primary Examiner Bergmeyer:Methods of Enzymatic Analysis," pp. 970, M, D. HENSLEY, AssistantExaminer 975, 983-4 (1965). 10

Dixon et al.: Enzymes, p. 337 (1964). US. Cl. X.R.

Chemical Abstracts, 50:10838c (1956). 19599

